Development of sensitive, universal diagnostic tools for fast and complete analysis of pathogen DNA is a critical priority in the Nation's biodefense effort. This proposal describes an advanced rapid sequencing by hybridization (rSBH) system designed to detect and identify any pathogen at the single organism level in complex biological samples. Based on similar technology being developed at Callida Genomics for fast, low cost human genome sequencing, the pathogen rSBH system would allow military or public health personnel rapid, inexpensive, and comprehensive information on the nature of pathogen infection before symptoms develop. In a typical rSBH experiment DNA fragments extracted from samples are interrogated with universal sets of labeled probes in a sequence-specific hybridization/ligation assay. An ultra-sensitive CCD camera detects positive ligation events, generating sequence information that is assembled computationally to determine complete sequences of all DNA present in the sample. Because the rSBH probe set is universal, containing all probes of a given length, it can detect and determine complete sequence information for trace quantities of pathogen DNA present in complex biological samples. This represents a significant advance over other DNA-based diagnostics, which require sequence-specific probes. The specific aims of the project are to develop and demonstrate a prototype rSBH pathogen diagnostics product by sequencing complete genomes and diagnostic genes of Yersinia pestis and Bacillus anthracis from cell culture and blood samples.